Depositing a binder on a solid support

ABSTRACT

A test device for use in determining analyte wherein binder is supported on a solid support in admixture with a marker whereby the presence and location of binder on the support can be determined prior to the assay. A control for the assay may also be applied to the support in admixture with a label.

This application is a continuation of application Ser. No. 106,075,filed Oct. 8, 1987, now abandoned.

FIELD OF THE INVENTION

This invention relates to a test device useful in an assay for ananalyte and to the use thereof in such assays. More particularly, thepresent invention relates to providing a binder for an analyte on asolid support for use in a solid phase assay for an analyte.

BACKGROUND OF THE INVENTION

Assays for determining analytes by a solid phase technique involvecontact between a sample suspected of containing the analyte and a testdevice having a binder specific for the analyte supported on a solidsupport. The amount of analyte which becomes bound to the binder on thesolid support is then detected with a tracer as a measure (quantitativeor qualitative) of analyte in the sample.

In some solid phase assays, for example, as described in U.S. Pat. No.4,632,901, the binder for the analyte is supported on a limited portionof the solid support in order to provide a test area and an areasurrounding the test area which is free of binder. The area free ofbinder may be employed as a background area to aid in determining ofanalyte by the use of a suitable tracer. When the test device includesmultiple layers, they need to be assembled with the test area orientedcorrectly relative to the other components of the device. Obtainingproper registry is difficult when the binder is not visible.

SUMMARY OF THE INVENTION

Use of a marker in admixture with a binder allows construction of testdevices where a test area of a solid support having binder on it isdistinguishable from a background area surrounding the test area. Thetest device of the present invention is for use in an assay for ananalyte. It has a solid support having a test area and a backgroundarea. A mixture of binder for the analyte and a marker is supported onthe test area. The test kit of the present invention includes the testdevice and a tracer having a tag which is distinguishable from themarker.

The assay of the present invention employs a test device having a solidsupport with a test area and a background area. The test device has amixture of a binder for an analyte and a marker supported on the testarea. The test device is contacted with a fluid sample and a tracer. Thetracer has a tag which is distinguishable from the marker.

In the manufacturing method of the present invention a solid supportwhich includes a test area and a background area is provided. A mixtureof binder and a marker is applied to the test area so that it issupported there. In this manner the marker is useful to determine thatbinder has in fact been applied to the test areas. The methodfacilitates assembly of the solid support with other components of thetest device without compromising performance of the binder under assayconditions.

The marker which is employed in admixture with the binder is one whichdoes not interfere with the binding properties of the binder, is capableof being supported on the solid support in a manner similar to thebinder, and does not interfere with the ability to detect tracer in thetest area.

In accordance with a preferred embodiment, the binder is supported onthe solid support by adsorption; accordingly, the marker which isemployed in admixture with the binder is one which is also capable ofbeing adsorbed by the solid support.

The preferred marker is a material which under appropriate conditions isdetectable with the naked eye. The marker may be a colored materialwhich absorbs light of a characteristic band within the visiblespectrum. Alternatively, and preferably, the marker is a fluorescentmaterial which upon excitation emits a characteristic fluorescent signalin the visible region of the spectrum. Colored and fluorescent materialsare referred to hereafter as "chromogens." With these chromogens,application of the mixture of binder and marker to the test area of thesolid support may be easily determined by the presence of color or uponexcitation by the presence of a fluorescent signal in the test area.

The marker which is preferably a chromogen, is applied to the solidsupport in admixture with the binder in an amount which does notadversely affect the binding properties of the binder, and which issufficient to permit detection of the marker in the test area. Themarker is preferably water soluble because in most cases a binder isapplied to a solid support in a water based solution.

Thus, by proceeding in accordance with the manufacturing method of thepresent invention, wherein a binder is applied to the test area of asolid support in admixture with a marker, a test area of the supporthaving binder securely attached it can be distinguished from abackground area. In this manner, if the solid support is to be locatedin a container which includes an inlet port for applying a sample andother materials to the test device, the test area in which the binder issupported can be properly located with respect to the sample inlet port.

In some cases, application of a control (for example an analyte positivecontrol) to a solid support is desirable. In accordance with anotheraspect of the present invention, the control (in particular a solutioncontaining a known amount of the analyte) is applied to a control areaof the solid support in admixture with a label, which is distinguishablefrom the marker whereby the control area may be determined by detectingthe label. In accordance with this aspect of the present invention, thecontrol area may be completely or partially overlapping with the testarea or it may be a completely separate area of the support. In mostcases, the control is a positive control; however, a negative controlmay also be applied.

Thus, in accordance with the present invention, the label which isapplied to the solid support in admixture with a control isdistinguishable from both the tag employed in the tracer used in theassay and from the marker applied in admixture with the binder. Thelabel which is applied in admixture with the control has thecharacteristics described above with respect to the marker; i.e., thematerial is one which does not interfere with the binding capabilitiesof the control, which is capable of being supported by the solid supportin a manner similar to the control, which does not interfere with theinteractions occurring under assay conditions, and which isdistinguishable from the tracer under assay conditions. The labelemployed in admixture with the control is also preferably a chromogenand most preferably a fluorescent material.

The present invention is useful in any assay where a binder is supportedon a solid support. The choice of binder is dependent on the assayformat and protocol. The selection of a suitable binder is deemed to bewithin the scope of those skilled in the art.

The solid support may take a wide variety of forms such as, for example,a sheet or membrane, a test strip, a dip stick, a card, or the like. Theselection of a suitable form is deemed to be within the scope of thoseskilled in the art. Similarly, the solid support may be made of a widervariety of materials. Preferably the support is porous.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top view of a test device which incorporates the presentinvention;

FIG. 2 is a side elevational view of the test device of FIG. 1;

FIG. 3 is a section taken along line 3--3 of FIG. 1; and

FIGS. 4A and 4B are simplified schematic representations of alternativeversions of first layer 11 which includes a binder and control.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a binder which is to beapplied to a test area of a solid support, preferably as an aqueoussolution or suspension is mixed with a marker, which is preferably achromogen, and in particular a fluorescent material. The mixture ofbinder and chromogen is then applied to a defined test area of the solidsupport, such as, for example, a spot, square, circle, triangle, or anyother shape desired, in a manner to support both the binder and thedetectable label in the test area. While those skilled in the art arefamiliar with a variety of coupling techniques to securely attach abinder to a solid support, adsorption is preferred. The procedure foradsorbing the binder can be any of those commonly available for patterncoating of materials including gravure printing, silk screening andother convenional printing methods.

The preferred materials for use as a marker are fluorescent materialswhich do not adversely affect the binding ability of the binder, andwhich are capable of being adsorbed by the support. As representativeexamples of suitable fluorescent materials, there may be mentionedAcridine Orange, Pyronin Y, Texas Red, Rhodamine 6G, with such materialsbeing employed in amounts which do not adversely affect the bindingability of the binder, which are detectable when subjected toultraviolet light, and which are not detectable with the naked eye. Ifthe material used as a marker includes groups which may be reactive withthe binder or control, such groups may be blocked prior to admixing thematerial, for example they may be placed in a buffer including a sourceof amino groups (e.g. glycine) that react with reactive sites on themarker and thereby prevent further reactivity which could influence thebiological activity of the binder or provide sites for non specificbinding under assay conditions.

After the binder has been supported on the solid support, the test areamay be distinguished from the background area by detecting the marker.Thus, for example, in the case where the label is a fluorescentmaterial, the support may be exposed to excitation energy of a suitablewave length (e.g. ultraviolet light from conventional U.V. lamps). Themarker will emit a characteristic fluorescent signal which is preferablydetectable with the naked eye.

In an embodiment employing a control, a solution or suspension of thecontrol and a label which is distinguishable from the marker employed isprepared, with the label preferably being a chromogen, and mostpreferably a fluorescent material. The solution or suspension of controland label is applied to the solid support in its designated controlarea, a portion of which or none of which may overlap with the testarea. The fact that the control has been applied to the solid support,and the area to which it has been applied, may be determined bydetecting the label, for example, in the case of a fluorescent materialby exposing the material to excitation energy of a suitable wavelength.

Thus, the binder may be applied to the solid support in admixture with afluorescent material having a characteristic emission band within thevisible spectrum and observable as a first color (such as yellow), andthe control may be applied in admixture with a fluorescent materialhaving a characteristic emission band within the visible spectrum andobservable as a second color (such as red), which is distinguishablefrom the first color. After application of both the control and thebinder, the solid support may be exposed to excitation energy ofsuitable wavelengths to detect the presence of both colors to therebydetermine the presence, in their respective areas, of both the binderand the control. Preferably the excitation wavelengths for the markerand the label are in the ultraviolet range of the sprectrum and in theabsence of U.V. irradiation, they are colorless or are colored with arelatively weak intensity.

The analyte may be determined (qualitatively or quantitatively) byapplying a sample suspected of containing the analyte and tracer to thetest area. The sample may be applied to an area smaller, equal to orgreater than the test area in which the binder is supported. Dependingon the chemistry of the assay, reactions take place and presence orabsence of a detectable signal from the tracer is indicative of thepresence of analyte.

While the chemistry of the assay is independent of the presentinvention, the preferred assay chemistry uses interactions amongspecific binding pairs to determine the presence of analyte. Thepreferred type of specific binding pairs are antigen/antibody pairs.

In the preferred assay chemistry, the tracer is one member of a specificbinding pair having a tag coupled to it. For example, if the assay is acompetitive format, the specific binding portion of the tracer would beone which is bound by the binder supported on the solid support. If theassay is a sandwich format, then the specific binding portion of thetracer would be bound by the analyte.

Such assay formats and amplification procedures are generally known inthe art, and a further description is not required for a completeunderstanding of the present invention.

The tag portion of the tracer may be any one of a wide variety ofmaterials, including, for example, enzymes, and chromogens. If the tagis detectable by color (as in colormetric enzyme tags and directdetection with a colored material) the color should be observable in thepresence of the marker and label. Presently preferred are coloredparticulate labels, such as liposomes including a chromogen. Theselection of a suitable tag is deemed to be within the scope of thoseskilled in the art.

In the assay technique, the tracer is applied to at least the test areaof the solid support, and in the case where a control is used, thetracer is also applied to at least the control area. The tracer may beapplied to an area greater than the test area and the control area.

In cases where a positive control is employed, the specific bindingportion of the tracer is bound by the positive control whereby the tagof the tracer should be detected in the control area. In addition, ifanalyte is present in the sample, the tag of the tracer should bedetected in the test area.

In accordance with a particularly preferred embodiment of the presentinvention, the binder in admixture with the marker is supported on atest area located at the surface of a solid support in a concentrationwhereby a tracer which includes a visible particulate label as its tagportion, under assay conditions, is visible on the support, withoutfurther treatment.

The binder supported on the test area is preferably present in at leastone microgram per cm², most generally at least 10 micrograms per cm²,and preferably 40 micrograms per cm². The residual binding capacity ofthe test area and the background area may be saturated or blocked bytreatment of the solid support with one or more types of proteins whichdo not specifically bind materials to be employed in the assay. Thus,for example, residual binding capacity may be blocked by use of bovineserum albumin. A wetting agent also may be applied to the test area.

In some cases, in applying the binder (in particular an antibody) to thetest area, a polyhydroxy compound (e.g., glycerol, erythritol, andsorbitol), or a sugar (e.g., glucose and sucrose) is included in theantibody solution to prevent non-specific binding (false positives)during the assay.

The solid support which is used is one which has a surface area(area/unit weight of material) such that the binder can be supported onthe support in a concentration (weight/unit area) such that the traceris visible under the assay conditions. The term "visible" as used hereinmeans that the material can be detected with the naked eye without theuse of instrumentation; although instrumentation may be used to detectthe intensity of the absorbence or fluorescence of the material.

The test area is preferably formed from a cellulose ester withnitrocellulose giving exceptionally good results. The term"nitrocellulose" refers to nitric acid esters of cellulose, which may benitrocellulose alone, or a mixed ester of nitric acid and other acids,and in particular, aliphatic carboxylic acids having from one to sevencarbon atoms, with acetic acid being preferred. Sheets which are formedfrom cellulose esterified with nitric acid alone, or a mixture of nitricacid and another acid such as acetic acid, are often referred to asnitrocellulose paper.

Although nitrocellulose is a preferred material for the test area (andthe entire solid support), other materials having a surface areasufficient for supporting the binder in a concentration as describedabove may also be employed.

As indicated above, in producing the preferred tracer one member of aspecific binding pair is labeled with a particulate label, which isvisible. A preferred particulate label is a sac, which includes a colorsubstance whereby the tracer, when used in the assay, is visible withoutdestruction of the sac to release the colored substance.

The sac which is used to label the specific binding portion of thetracer may be any one of a wide variety of sacs, including but notlimited to liposomes (single walled or multilamellar) or polymermicrocapsules (for example, those made by coascervation, or interfacialpolymerization).

Polymer microcapsules also may be produced by procedures known in theart except that the solution in which the microcapsules are formed alsoincludes the tag whereby the interior of the polymer microcapsuleincludes the tag. The preparation of such microcapsules is disclosed forexample in Microencapsulation Processes and Applications, edited by JanE. Vandegger (Plenum Press, 1974). polymer nucleus coated with such adye or pigment. Such labels are described in more detail in U.S. Pat.No. 4,373,932, which is hereby incorporated by reference. The tracersproduced in accordance with that patent may also be employed as tracersin the present invention.

The visible particulate label may be visible polymer particles, such ascolored polystryrene particles, preferably of spherical shape.

Representative examples of other suitable particulate labels includeferritin, phycoerythrins or other phycobili-proteins; precipitated orinsoluble metals or alloys; fungal, algal, or bacterial pigments orderivatives such as bacterial chlorophylls; plant materials orderivatives, and the like.

The specific binding portion of the tracer may be labeled with theparticulate label so as to produce a tracer for use in the invention byprocedures generally known in the art, with the procedure which is usedbeing dependent upon the choice of specific binding species and theparticulate label. Such techniques include covalent coupling,derivatization or activation, and the like. In producing a tracerwherein the binder is labeled with a sac, the sac may be produced from acomponent which has been derivatized with a specific binding species,whereby the sac, when produced, is coupled to the specific bindingportion.

Thus, the preferred tracer is comprised of a specific binding portionand a particulate label (solid or solid-like, as opposed to non-solid

As known in the art, liposomes can be prepared from a wide variety oflipids, including phospholipids, glycolipids, steroids, relatively longchain alkyl esters; e.g., alkyl phosphates, fatty acid esters; e.g.,leitchin, fatty amines and the like. A mixture of fatty materials may beemployed, such as a combination of neutral steroid, a charged amphiphileand a phospholipid. As illustrative examples of phospholipids, there maybe mentioned lecithin, sphingomyelin, dipalmitoyl, and the like. Asrepresentative steroids, there may be mentioned cholesterol,cholestanol, lanesterol, and the like. As representative examples ofcharged amphiphilic compounds, which generally contain from 12 to 30carbon atoms, there may be mentioned mono- or dialkyl phosphate ester oran alkylamine; e.g., dicetyl phosphate, stearyl amine, hexadecyl amine,dilauryl phosphate, and the like.

The liposome sacs are prepared in an aqueous solution including the tagwhereby the sacs will include the tag in their interior. The liposomesacs may be prepared by vigorous agitation in the solution, followed byremoval of tag from the exterior of the sac.

Further details with respect to the preparation of liposomes are setforth in U.S. Pat. No. 4,342,826 and PCT International Publication No.WO80/01515, both of which are hereby incorporated by reference.

The tracer may also be produced by labeling the specific binding specieswith an aqueous dispersion of a hydrophobic dye or pigment, or a labels,such as radioisotopes, enzymes and various fluorescent materials), andthe particulate label provides a tag which is visible under the assayconditions so that the presence or amount of analyte may be determinedwithout further treatment and without the use of instrumentation; e.g.,by the use of a liposome containing a colored material as theparticulate label.

In accordance with a preferred embodiment, the test device isconstructed as described in commonly assigned U.S. Ser. No. 016,846, nowabandoned and Ser. No. 106,757, filed Aug. 8, 1987. The preferred deviceincludes a porous solid support for supporting the binder at a test areain a concentration whereby the tracer used in the assay, when bound tothe test area, under assay conditions is visible without furthertreatment. It also includes a flow control layer, beneath the poroussolid support, which is formed of a porous material having a pore sizeto control the rate of flow of assay reagents through the test area. Thetest device also preferably includes a porous spacer layer for spacingan absorbent layer, formed of an absorbent material, from the flowcontrol layer.

The absorbent layer has an absorbency sufficient to absorb the reagentliquids applied to the test layer during the assay. In addition, theabsorbent materials functions to provide for flow through the testlayer.

The entire porous solid support may be formed of the material used inthe test area. Alternatively, only the test area may be formed of such amaterial.

In addition, since the test device is employed in a manner such that theassay reagents flow through its layers, solid support has a pore sizewhich is greater than the size of the particulate label employed in theassay so that portions of the tracer, which do not become bound underassay conditions, flow into the absorbent layer and are not visible atthe test area. In general, the solid support should have a pore sizewhich is at least 2 um, and most preferably at least 5 um. In general,the pore size does not exceed 12 um. It is to be understood, however,that although the previously described pore sizes are preferred, otherpore sizes may be employed, depending upon the materials used in theassay.

The flow control layer of the test device is formed of a porous materialwhich is employed to control the rate of flow of assay reagents throughthe test layer and into the absorbent layer. The preferred porousmaterial which is employed in forming the flow control layer has a poresize which is less than the pore size of the material employed forforming the solid support. Thus, in effect, the flow control layerfunctions to reduce the rate of flow of assay reagents through the moreporous test area.

The pore size of the flow control layer, as well as the thickness of theflow control layer, is preferably controlled in a manner such that theflow of assay reagents through the test area provides the requisitesensitivity as well as a rapid and accurate assay.

In accordance with one particularly preferred embodiment, the layer forcontrolling rate of flow through the test device is dimensioned andsized in a manner such that the flow rate of materials through the testarea is in the order of at least 0.5 ml/min, and generally no more than2 ml/min.

The flow control layer is preferably formed from a non-fibrous materialpolycarbonate and having pores or channels of a uniform size thatprovide for unidirectional flow from the test layer to the layer beneaththe flow control layer.

Immediately, below the flow control layer of the preferred test device,a spacer layer is provided. It is a porous material which functions as aspacer between the flow control layer, and the absorbent layer. Theporous spacer layer has a pore size greater than the pore size of theflow controlling layer so that the spacer layer does not function torestrict flow through the test device.

The preferred test device also includes an absorbent layer which is aporous material having an absorbing or absorbent capacity sufficient toabsorb the liquids which flow into the test device during the assay. Theabsorbent layer also functions to provide a driving force (e.g., aconcentration differential) which causes reagents applied to the testarea to flow into the absorbent layer.

Thus, in accordance with the preferred embodiment of the presentinvention, an assay employs a tracer wherein the tag portion of thetracer is a visible particulate sac, and wherein the assay is performedon a test device, which is preferably formed from a plurality of layersof material having different characteristics, as described above, andwherein the assay reagents flow through the test area of the testdevice.

The materials which are employed in forming the various layers of thetest device are selected to have the characteristics described above. Inaddition, such materials should not produce non-specific binding ofanalyte or tracer. The materials may inherently have suchcharacteristics, or alternatively, the materials may be treated toprevent nonspecific binding; for example, treatment with an appropriateprotein, such as bovine serum albumin. The solid support of the testdevice is preferably also treated with a wetting agent in order toinsure proper flow of the assay reagents through the test layer and intothe absorbent layer. Representative examples of wetting agents includesucrose, glycerol, glucose, and sorbitol. The solid support may besimultaneously treated with a protein and wetting agent; e.g., anaqueous solution of bovine serum albumin and sucrose.

In general, the test device is mounted on or in a suitable holder, suchas a card or a container. The selection of a suitable holder for thetest device is deemed to be within the scope of those skilled in theart.

In addition, the preferred test device is provided with a cover havingan aperature, which directs assay reagents to the test area. Thus, forexample, the test device may be covered with a card including anaperture which overlies the test area whereby the liquid sample andvarious assay reagents are applied directly to the test area.Alternatively, the test device may be placed in a container whichincludes a suitable aperture for directing the sample and assay reagentsto the test area. The use of a marker in admixture with the binderidentifies the test area whereby it may be properly positioned withrespect to the aperture.

Referring now to the drawings, a test device, generally designated as10, comprised of a solid support, generally designated as 11, which hasa test area for supporting a binder. A preferred material isnitrocellulose, which has a pore size in excess of 2 microns, andgenerally less than 12 microns. Most preferred is a pore size of about 5microns.

Immediately underneath the layer 11 is a flow control layer 12 which ispreferably formed from a unidirectional flow controlling polycarbonatemembrane having a pore size of 0.6 microns.

Immediately underneath flow controlling layer 12 a spacer layer 13 isprovided. The spacer layer 13 is formed of a porous material, andgenerally has a pore size greater than the pore size of flow controllinglayer 12. The layer 13 may be formed, for example, from a non-wovenpolyacetate.

Immediately underneath spacer layer 13 and in contact therewith isabsorbent layer 14. The absorbent layer 14 is preferably formed from acellulose material, e.g., absorbent cellulose paper.

Thus, the test device is comprised of layers 11, 12, 13 and 14, whichare preferably combined to produce a unified device 10. The layers maybe attached to each other, for example, by sewing of the layers to eachother; however, other methods of attachment are possible.

As particularly shown, the test device 10 is in a test container whichincludes a base 15, and a cover 16. The base 15 has a depth such thatthe device 10 is within the container. In the preferred embodiment thecontainer has three sides in a generally triangular shape with roundedcorners.

The cover 16 includes a raised portion having a suitable aperture 17which overlies the test area 18 of the solid support 11. Preferably, aportion of the background area of the support 11 surrounding the testarea is also within the opening defined by aperature 17.

The cover 16 is supported over layer 11 by projections 19 extendingupward from the sides of the base 15. The projections 19 are ofsufficient height so as to provide air spaces 20 which provide forventilation of the sides of the device 10. The air spaces 20 are boundedby the projections 19, the cover 16, and the base 15.

The raised portion of the cover 16 surrounding the aperture 17 include acolored area 21, the color of which preferably contrasts from that ofthe cover 16 and the color to be generated in test area 18 to providefor a better reading of the test results which are generally determinedby color. In the preferred embodiment, base 15, cover 16, and coloredarea 21 are made of plastic materials.

For use of the test device 10 in a sandwich fluorescent signal whenexposed to ultraviolet energy.

In manufacturing the test article, solid support 11 is initially treatedwith a mixture of binder and marker which may be a first fluorescentmaterial to apply the mixture to test area 101. Thereafter, a mixture ofthe control and label which may be a second fluorescent material isapplied to the control area 102. As shown, control area 102 is smallerand lies within area 101.

The fact that the analyte and binder have been deposited in theirrespective areas may then be determined by exposing solid support 11 toultraviolet light and determining the presence of the respectivefluorescent emission colors in the appropriate areas.

Thereafter, solid support 11 may be combined with the other layers, andthe device 10 positioned in the test container in a manner such that thetest area 101 is beneath aperture 17, whereby assay reagents may bedirected to the area in which the binder and control is supported.

An alternative embodiment is shown in FIG. 4B wherein control area 102'is where the control is deposited and test area 101' is where the binderis deposited. As should be apparent, the presence of the control and thebinder in their respective areas may be determined as described withreference to FIG. 4A.

In the embodiment of FIG. 4B, if no analyte is present, only area 102'is colored by the tracer, thereby indicating a negative sign on thelayer 11. If analyte is present in the sample, assay, the binder may bean antibody specific for the analyte to be determined. If the sandwichassay is to be operated in a sequential mode a sample which is suspectedof containing the analyte is applied to the test area through theaperture 17 whereby the sample contacts the binder in test area 18, withthe sample flowing through the test device to the absorbent layer 14.The analyte present in the sample will become specifically bound to thebinder in area 18.

Thereafter, tracer is applied to the test device through the aperture17. The tracer becomes bound to the analyte, and any unbound portionflows through the test device to the absorbent layer 14.

If desired, a wash solution may be applied to the test device 10 priorto addition of the tracer. Similarly, after addition of the tracer, awash solution may be applied to the test device to wash any tracer whichmay not be specifically bound to the complex in area 18, into theabsorbent layer 14.

The presence of color in area 18 is indicative of the presence ofanalyte, and if the assay is to be a quantitative assay, the intensityof such color is indicative of the quantity of analyte present in thesample.

Referring now to FIG. 4A solid substrate 11 includes a test area 101 andcontrol area 102. Test area 101 includes a mixture of the binder used inthe assay and a marker, such as a fluorescent material. Control area 102includes an analyte control in admixture with a label, preferably asecond fluorescent material, which emits a both areas 101' and 102' willbe colored, whereby a plus sign is displayed, indicating the presence ofanalyte in the sample.

Although the invention has been described with respect to a preferredembodiment as shown in the drawings, the present invention is equallyapplicable to depositing binder, and optionally also control on a solidsupport other than one particularly shown. Thus, for example, the testdevice may be in the form of a single layer, or two layers. Similarly,the test layer may be other than a membrane as shown.

These and other modifications should be apparent to those skilled in theart.

EXAMPLE

A sulfonyl chloride derivative of sulforhodamine 101 (available fromMolecular Probes Cat. T353 as Texas Red Dye), after having beenincubated for two hours in a 0.1 M glycine buffer pH 8.0 is combinedwith rabbit anti-group A Streptococcus antibody (n-acetyl glucosamineaffinity purified) in a concentration ranging from 0.5 to 20 ug/ml.Separately Group A Streptococcus antigen is extracted with nitrous acid(300 ul HCl (0.1 M) with 40 ul NaNO₂ (4M) and after three minutes 40 ulTris buffer (1M, Trizma™ base (Sigma), with 4M NaCl)is added). To theextracted antigen is added Rhodamine 6G dye (Allied Cat. 663) in glycinebuffer (0.1M) pH 8.0 to provide a concentration of less than 4 ug/ml.

Each of the above solutions is applied to nitrocellulose membrane(Schleicher & Schuell, pore size 5 microns) and under long wave (290-350nm) ultraviolet excitation, the Texas Red, which fluoresces red, may bedistinguished from the Rhodamine 6G which fluoresces bright yellow. Thepresence of the two colors discriminates and identifies the location ofthe antibody binder and antigen positive control.

The nitrocellulose containing the mixture of antibody and fluorescentmaterial as well as the mixture of antigen positive control andfluorescent material is used in a sandwich assay for detecting Group AStrep antigen in which the tracer is affinity purified antibody (rabbitanti-group A Strep) covalently coupled to liposomes containingsulfo-rhodamine B dye (the procedure for making this tracer is describedin South Africa Patent No. 84/9397 corresponding to allowed U.S. Ser.No. 579,667 which is hereby incorporated by reference).

The presence of the fluorescent material with the antibody binder andwith the antigen positive control on the nitrocellulose support does notadversely affect sensitivity at 5×10⁵ organisms/ml.

The present invention is applicable to procedures and products fordetermining a wide variety of analytes. As representative examples oftypes of analytes, there may be mentioned; drugs, including therapeuticdrugs and drugs of abuse; hormones, vitamins, proteins, includingantibodies of all classes; peptides; steroids; bacteria; fungi; viruses;parasites; components or products of bacteria, fungi, viruses, orparasites; allergens of all types; and products or components of normalor malignant cells. As particular examples, there may be mentioned T₄ ;T₃ ; digoxin; hCG; insulin; theophylline; luteinizing hormone; organismscausing or associated with various disease states, such as StreptococcusPyogenes (group A), Herpes Simplex I and II, cytomegalovirus, rubella,chlamydia, and Candida Albicans.

The analyte may be determined in various samples, including, forexample, body fluids, such as saliva, urine, serum, and cerebral spinalfluid or from swab samples, e.g., from the throat.

Numerous modifications and variations of the present invention arepossible in light of the above, and therefore, within the scope of theappended claims, the invention may be practiced otherwise than asparticularly described.

What is claimed is:
 1. A test device for use in detecting the presenceof an analyte, comprising:a casing having an aperture; a solid supportcomprising an upper surface and contained in said casing; a test area onthe upper surface of said support and positioned in registry with saidaperture; a binder for said analyte attached to said test area; adetectable chromogen marker attached to said binder, related to thepresence or absence of the binder which does not interfere with saidassay; and a background area on the upper surface of said supportwithout binder and detectable marker; wherein: said marker causes thebinder to be distinguishable from said support so that, before thedevice is used in said assay the device is examined to confirm thatbinder is present in said test area.
 2. A test device for use in anassay for detecting the presence of an analyte, comprising:a solidsupport comprising an upper surface and a lower surface; a test area onthe upper surface of said support; a binder for the analyte attached tosaid test area; a detectable marker attached to said binder which isrelated to the presence or absence of the binder, and does not interferewith said assay; and a background area on the upper surface of saidsupport without binder and detectable marker; a control area on theupper surface of said support; an assay control attached to said controlarea; and a detectable label attached to said assay control which isdistinguishable and does not interfere with the assay or with thedetectable marker; wherein: the detectable marker causes the binder tobe distinguishable from said support so that, before the device is usedin said assay the device is examined to confirm that binder is presentin said test area; and said label causes the assay control to bedistinguishable from said support so that before the device is used insaid assay the device is examined to confirm that assay control ispresent on said support.
 3. The test device of claim 2 wherein the assaycontrol is a positive analyte control.
 4. The test device of claim 3wherein said control area is attached to said test area.
 5. The testdevice of claim 2 wherein the label is a chromogen.
 6. The test deviceof claim 5 wherein the solid support is nitrocellulose.
 7. A process forproducing a test device suitable for detecting the presence of ananalyte, comprising:providing a solid support comprising an uppersurface; contacting said upper surface with a binder for said analyte;contacting said binder with a detectable marker related to the presenceor absence of the binder which does not interfere with said assay;detecting the marker to confirm that binder is present in said device;and placing the support in a casing including an aperture with the testarea in registry with the aperture.
 8. The process of claim 7 furthercomprising:contacting said upper surface with a mixture of an assaycontrol; and contacting said assay control with a detectable label;wherein: said control area is distinguishable from said test area andsaid support, and said label is related to the presence or absence ofsaid assay control.
 9. The process of claim 8 wherein said control areais attached to said test area.
 10. The process of claim 8 wherein themarker is a fluorescent material and the label is a differentfluorescent material.
 11. The process of claim 7 wherein the marker is achromogen.
 12. The process of claim 7 wherein the marker is afluorescent dye.
 13. The process of claim 7 wherein the support isnitrocellulose.